The proposed research aims to extend and refine the mechanisms utilized by catalytic antibodies. The proposal focuses on the in vitro evolution of synthetic human antibodies to perform defined catalytic tasks. This research will replace the use of animals in the production of antibodies. In preliminary experiments we have demonstrated the ability to construct and select functional synthetic human antibodies from vast phage display libraries. In vivo chain shuffling is proposed as a route to facilitate directed molecular evolution of antibodies using iterative selection strategies. Selections will be performed from the vast collection of synthetic antibodies we have created with a series of haptens designed to refine or recruit a catalytic mechanism. In vivo chain shuffling will allow for the automatic introduction of diversity into the pool of primary selected antibodies. The diversified pool will then be reselected to enhance the catalytic function of the antibodies. Libraries designed for the selection of antibodies which catalyze acyl transfer reactions will be constructed based on our knowledge of the structure and sequence of known catalytic antibodies. Using these strategies we will select and evolve the function of antibodies which use metal cofactors to assist in catalysis. This work is supported by our preliminary data demonstrating the ability to directly select antibodies which ligate metals. We will extend the repertoire of mechanisms utilized by catalytic antibodies by building on our work with chemical-event selections. These selections will evolve antibodies to catalyze reactions by mechanisms programmed by the experimenter. A major focus of these different strategies is the evolution of sequence specific proteases. The proposed work will result in catalytic antibodies which mimic the catalytic mechanisms of metallo- and cysteine and serine proteases. The ultimate goal is the construction of human antibodies which catalytically neutralize viruses by sequence specific hydrolysis of their coat proteins. These catalysts may also be of use in the fragment coupling of proteins and serve to complement traditional synthetic methods. Directed evolution will also be developed for the selection of antibody aldolases. These catalysts will be of use in the synthesis of enantiomerically pure drugs. To assess our ability to evolve antibodies in a directed fashion, catalysts will be studied as a function of sequence and kinetic mechanism. In collaboration with projects by Wright Wilson structural studies will be performed which will allow comparison of laboratory evolved catalysis with natural enzymes.